JR209TA cloned experiment

Ta cloned experiment

Method (TA cloning): a method of connecting a PCR fragment to a carrier DNA having 3-T protruding. Because the polymerase applied in the PCR reaction has the activity of end metastasis, usually in 3 plus A. For example: TAQ polymerase simultaneously has a function of end linase, and a 3-A protruding end is automatically added at 3 ends of each PCR amplification product when the PCR reaction is reacted. Connection can only be paired by T / A pairing only with a fragment of a DNA having a 3-T protruding end.

During the presence of the PCR, the purpose of the target gene is often unclear, the discouragement of the target gene is often required to be cloned by TA. The method, recombinant into the T-vector, and the DNA sequence is determined by sequence. Due to the different DNA polymerases used in the PCR, it is often divided into two cases: (1) use different TAQ enzymes, after the end of the PCR amplification cycle, plus 72 ° C for 10 minutes, TAQ enzyme can be The 3 terminals of the amplified product plus A, so the PCR product can be directly connected to the T vector after recovering purification. (2) Using high-fidelity DNA polymerase, such as PFU enzymes, since it cannot be plus A, the obtained DNA sequence is blunt, and therefore, it is necessary to add a process after recovery purification. Usually, the PCR recovery product is a template, plus the amount of ordinary TAQ enzyme and the reaction solution, adding DATP (or DNTP), 72 ° C for 10 minutes, and then adding the A product directly for TA connections.

Experimental material requirements: Unpurified PCR products and PCR electrophoresis pictures, PCR amplification primer sequence PCR amplification enzyme, if used in 3 u0026 # 39; u0026 Rarr; 5 u0026 # 39; External enzyme activity polymerase, please provide PCR primers, we will re-amplify the PCR product to operate TA clones. Service Description

1, PCR product TA cloning experiment, the larger the PCR fragment, the larger the difficulty of TA clone; 2, TA clone experiment In, we use the common T vector as a standard T vector, if there is a special requirement for the carrier, please inform the customer service person before experiment.

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